T cell receptors

ABSTRACT

The present invention relates to T cell receptors (TCRs) which bind the HLA-A*0201 restricted peptide GVYDGEEHSV (SEQ ID NO: 1) derived from the MAGE-B2 protein. The TCRs of the invention demonstrate excellent specificity profiles for this MAGE epitope. Also provided are nucleic acids encoding the TCRs, cells engineered to present the TCRs, cells harbouring expression vectors encoding the TCRs and pharmaceutical compositions comprising the TCRs, nucleic acids or cells of the invention.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of application Ser. No. 17/501,200filed Oct. 14, 2021, which is a continuation of application Ser. No.16/154,270 filed Oct. 8, 2018, now abandoned, which is acontinuation-in-part application of international patent applicationSerial No. PCT/EP2017/058578 filed Apr. 10, 2017, which published as PCTPublication No. WO 2017/174823 on Oct. 12, 2017, which claims benefit ofGB patent application Serial No. 1606156.6 filed Apr. 8, 2016.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appin cited documents”) and all documents cited orreferenced in the appin cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (344045.xml; Size:54,829 bytes; and Date of Creation: Aug. 1, 2023) is herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to T cell receptors (TCRs) which bind theHLA-A*0201 restricted decapeptide GVYDGEEHSV (SEQ ID NO: 2) derived fromthe melanoma-associated antigen (MAGE) B2 protein (amino acids 230-239),and the HLA-A*0201 restricted decapeptide GVYDGREHTV (SEQ ID NO: 1)derived from the melanoma-associated antigen (MAGE) A4 protein. TCRs ofthe invention demonstrate excellent specificity profiles for these MAGEepitopes.

BACKGROUND TO THE INVENTION

Cancer testis antigens (CTA) are a subclass of tumour-associated antigen(TAA) encoded by approximately 140 genes. Expression of these antigensis restricted in immune privileged sites such as the testes, placentaand fetal ovary; they are typically not expressed in other tissues.Expression of these genes has been observed in malignant tumors. Theimmunogenicity of CTA has led to the widespread development of cancervaccines targeting these antigens in many solid tumors. Within thislarge class of TAA, melanoma-associated antigens (MAGE) have emerged aspromising candidates for cancer immunotherapy.w

More than 30 cancer testis (CT) genes have been reported as members ofmulti-gene families that are organized into gene clusters on chromosomeX (CT-X antigens). The CT gene clusters are located between Xq24 andXq28 and include gene families such as MAGE and NY-ESO-1. Type I MAGEgene clusters are the most extensively characterized and include theMAGE-A, MAGE-B and MAGE-C families. The MAGE-A proteins are encoded by12 different MAGE-A gene family members (MAGE-A1 to MAGE-A12) and aredefined by a conserved 165-171 amino acid base, called the MAGE homologydomain (MHD). The MHD corresponds to the only region of shared aminoacids by all of the MAGE-A family members.

T cells recognize and interact with complexes of cell surface molecules,referred to as human leukocyte antigens (“HLA”), or majorhistocompatibility complexes (“MHCs”), and peptides. The peptides arederived from larger molecules, which are processed by the cells whichalso present the HLA/MHC molecule. The interaction of T cells andHLA/peptide complexes is restricted, requiring a T cell specific for aparticular combination of an HLA molecule and a peptide. If a specific Tcell is not present, there is no T cell response even if its partnercomplex is present. Similarly, there is no response if the specificcomplex is absent, but the T cell is present. The mechanism is involvedin the immune system's response to infection, in autoimmune disease, andin responses to abnormalities such as tumours.

Some MAGE gene family proteins are only expressed in germ cells andcancer (MAGE-A to MAGE-C families). Others are widely expressed innormal tissues (MAGE-D through to MAGE-H). All these MAGE proteinfamilies have a homologous region that is closely matched to thesequence of the other MAGE proteins and comprises peptides displayed asHLA/peptide complexes in immune recognition. Hence, it is important toselect TCR clinical candidates that are highly specific for the desiredMAGE peptide/HLA-A2 antigen.

MAGE B2 is a CTA member of the MAGE B gene family. The function isunknown, though it is thought that it may play a role in embryonaldevelopment. In tumour pathogenesis, it appears to be involved in tumortransformation or aspects of tumor progression. MAGE B2 has beenimplicated in a large number of tumours, The peptide GVYDGEEHSV (SEQ IDNO: 2) corresponds to amino acid residue numbers 231-241 of the knownMAGE-B2 protein.

MAGE A4 is a CTA of the MAGE A gene family. MAGE A4 is expressed intestis and placenta, and in a significant fraction of tumors of varioushistological types. The peptide GVYDGREHTV (SEQ ID NO: 1) showscross-reactivity with MAGE B2, such that certain TCRs are able to bindto HLA molecules displaying both peptides.

SUMMARY OF THE INVENTION

We have developed a TCR which binds to HLA molecules displaying the MAGEB2 peptide GVYDGEEHSV (SEQ ID NO: 2) as well to MAGE A4 peptideGVYDGREHTV (SEQ ID NO: 1). In a first aspect, the present inventionprovides a T cell receptor (TCR) having the property of binding toGVYDGEEHSV (SEQ ID NO: 2) and GVYDGREHTV (SEQ ID NO: 1) in complex withHLA-A*0201 with a dissociation constant of from about 0.05 μM to about20.0 μM when measured with surface plasmon resonance at 25° C. and at apH between 7.1 and 7.5 using a soluble form of the TCR, wherein the TCRcomprises a TCR alpha chain variable domain and a TCR beta chainvariable domain, and wherein the TCR variable domains form contacts withat least residues V2, Y3 and D4 of GVYDGEEHSV (SEQ ID NO: 2). and withat least residues V2, Y3 and D4 of GVYDGREHTV (SEQ ID NO: 1)

In embodiments, the TCR according to the invention has the property ofbinding to GVYDGEEHSV (SEQ ID NO: 2) and GVYDGREHTV (SEQ ID NO: 1) incomplex with HLA-A*0201 with a dissociation constant of from about 20 μMto about 50 μM when measured with surface plasmon resonance at 25° C.and at a pH between 7.1 and 7.5 using a soluble form of the TCR, whereinthe TCR comprises a TCR alpha chain variable domain and a TCR beta chainvariable domain. In some embodiments, the dissociation constant is above50 microM, such as 100 μM, 200 μM, 500 μM or more.

In some embodiments, the alpha chain variable domain of the TCRcomprises an amino acid sequence that has at least 80% identity to thesequence of amino acid residues 1-105 of SEQ ID NO: 3 (alpha chain),and/or the beta chain variable domain comprises an amino acid sequencethat has at least 80% identity to the sequence of amino acid residues1-123 of SEQ ID NO: 4 (beta chain).

In a further aspect, the present invention provides a T cell receptor(TCR) having the property of binding to GVYDGEEHSV (SEQ ID NO: 2) andGVYDGREHTV (SEQ ID NO: 1) in complex with HLA-A*0201 and comprising aTCR alpha chain variable domain and a TCR beta chain variable domain,

-   -   the alpha chain variable domain comprising an amino acid        sequence that has at least 80% identity to the sequence of amino        acid residues 1-105 of SEQ ID NO: 3, and/or    -   the beta chain variable domain comprising an amino acid sequence        that has at least 80% identity to the sequence of amino acid        residues 1-123 of SEQ ID NO: 4.

The GVYDGEEHSV (SEQ ID NO: 2) HLA-A2 and GVYDGREHTV (SEQ ID NO: 1)HLA-A2 complexes provide cancer markers that the TCRs of the inventioncan target. The present invention provides such TCRs useful for thepurpose of delivering cytotoxic or immune effector agents to the cancercells and/or useful for use in adoptive therapy.

TCRs are described using the International Immunogenetics (IMGT) TCRnomenclature, and links to the IMGT public database of TCR sequences.Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Broadly, each chain comprises variable, joining and constantregions, and the beta chain also usually contains a short diversityregion between the variable and joining regions, but this diversityregion is often considered as part of the joining region. Each variableregion comprises three CDRs (Complementarity Determining Regions)embedded in a framework sequence, one being the hypervariable regionnamed CDR3. There are several types of alpha chain variable (Vα) regionsand several types of beta chain variable (Vβ) regions distinguished bytheir framework, CDR1 and CDR2 sequences, and by a partly defined CDR3sequence. The Vα types are referred to in IMGT nomenclature by a uniqueTRAV number. Thus “TRAV21” defines a TCR Vα region having uniqueframework and CDR1 and CDR2 sequences, and a CDR3 sequence which ispartly defined by an amino acid sequence which is preserved from TCR toTCR but which also includes an amino acid sequence which varies from TCRto TCR. In the same way, “TRBV5-1” defines a TCR Vβ region having uniqueframework and CDR1 and CDR2 sequences, but with only a partly definedCDR3 sequence.

The joining regions of the TCR are similarly defined by the unique IMGTTRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRACand TRBC nomenclature.

The beta chain diversity region is referred to in IMGT nomenclature bythe abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJregions are often considered together as the joining region.

The α and β chains of αβ TCR's are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region and joining region. Inthe present specification and claims, the term “TCR alpha variabledomain” therefore refers to the concatenation of TRAV and TRAJ regions,and the term TCR alpha constant domain refers to the extracellular TRACregion, or to a C-terminal truncated TRAC sequence. Likewise the term“TCR beta variable domain” refers to the concatenation of TRBV andTRBD/TRBJ regions, and the term TCR beta constant domain refers to theextracellular TRBC region, or to a C-terminal truncated TRBC sequence.

The unique sequences defined by the IMGT nomenclature are widely knownand accessible to those working in the TCR field. For example, they canbe found in the IMGT public database. The “T cell Receptor Factsbook”,(2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 alsodiscloses sequences defined by the IMGT nomenclature, but because of itspublication date and consequent time-lag, the information thereinsometimes needs to be confirmed by reference to the IMGT database.

One TCR in accordance with the invention comprises an alpha chainextracellular domain as shown in SEQ ID NO: 3 (TRAV10+TRAC) and a betachain extracellular domain as shown in SEQ ID NO: 4 (TRBV24-1+TRBC-2).The terms “parental TCR”, “parental MAGE-A4 TCR”, are used synonymouslyherein to refer to this TCR comprising the extracellular alpha and betachain of SEQ ID NOs: 2 and 3 respectively. It is desirable to provideTCRs that are mutated or modified relative to the parental TCR that havea higher affinity and/or a slower off-rate for the peptide-HLA complexthan the parental TCR.

For the purpose of providing a reference TCR against which the bindingprofile of such mutated or modified TCRs may be compared, it isconvenient to use a soluble TCR in accordance with the invention havingthe extracellular sequence of the parental MAGE-A4 TCR alpha chain givenin SEQ ID NO: 3 and the extracellular sequence of the parental MAGE-A4TCR beta chain given in SEQ ID NO: 4. That TCR is referred to herein asthe “the reference TCR” or “the reference MAGE-A4 TCR”.

TCRs of the invention may be non-naturally occurring and/or purifiedand/or engineered. TCRs of the invention may have more than one mutationpresent in the alpha chain variable domain and/or the beta chainvariable domain relative to the parental TCR. “Engineered TCR” and“mutant TCR” are used synonymously herein and generally mean a TCR whichhas one or more mutations introduced relative to the parental TCR, inparticular in the alpha chain variable domain and/or the beta chainvariable domain thereof. These mutation(s) may improve the bindingaffinity for GVYDGEEHSV (SEQ ID NO: 2) in complex with HLA-A*020101. Incertain embodiments, there are 1, 2, 3, 4, 5, 6, 7 or 8 mutations inalpha chain variable domain, for example 4 or 8 mutations, and/or 1, 2,3, 4 or 5 mutations in the beta chain variable domain, for example 5mutations. In some embodiments, the α chain variable domain of the TCRof the invention may comprise an amino acid sequence that has at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% identityto the sequence of amino acid residues 1-105 of SEQ ID NO: 3. In someembodiments, the (3 chain variable domain of the TCR of the inventionmay comprise an amino acid sequence that has at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to the sequence ofamino acid residues 1-123 of SEQ ID NO: 4.

The alpha chain variable domain of a TCR of the invention may have thefollowing mutation:

CDR2 M4 V CDR3 S4 T CDR3 S4 Nwith reference to the numbering shown in SEQ ID NO: 3, and/or

-   -   the beta chain variable domain may have at least one of the        following mutations:

CDR3 N10 G CDR3 N10 V CDR2 S1 A CDR3 S4 T CDR3 N10 Ewith reference to the numbering shown in SEQ ID NO: 4.

The alpha chain variable domain of a TCR of the invention may comprisethe amino acid sequence of amino acid residues 1-105 of SEQ ID NO: 3 or7 or 8 or 9

-   -   or an amino acid sequence in which amino acid residues 1-27,        34-47, and 54-90 thereof have at least 90% or 95% identity to        the sequence of amino acid residues 1-27, 34-47, and 54-90        respectively of SEQ ID NO: 3 or 7 or 8 and in which amino acid        residues 28-33, 48-53 and 91-105 have at least 90% or 95%        identity to the sequence of amino acid residues 28-33, 48-53 and        91-105 respectively of SEQ ID NO: 3 or 7 or 8 or 9.

In the alpha chain variable domain, the sequence of

-   -   (i) amino acid residues 1-27 thereof may have (a) at least 90%        identity to the sequence of amino acid residues 1-27 of SEQ ID        NO: 3 or (b) may have one, two or three amino acid residues        inserted or deleted relative to the sequence of (a);    -   (ii) amino acid residues 28-33 of SEQ ID NO: 3;    -   (iii) amino acid residues 34-47 thereof may have (a) at least        90% identity to the sequence of amino acid residues 34-47 of SEQ        ID NO: 3 or (b) may have one, two or three amino acid residues        inserted or deleted relative to the sequence of (a);    -   (iv) amino acid residues 48-53 of SEQ ID NO: 3 or amino acid        residues 48, 49 and 51-53 of SEQ ID NO: 3 with amino acid        residue 50 of SEQ ID NO: 3 substituted with R in place of 1;    -   (v) amino acid residues 54-90 thereof may have at least 90%        identity to the sequence of amino acid residues 54-90 of SEQ ID        NO: 3 or may have one, two or three insertions, deletions or        substitutions relative thereto;    -   (vi) amino acid residues 91-105 of SEQ ID NO: 3.

The beta chain variable domain of a TCR of the invention may comprisethe amino acid sequence of SEQ ID NO: 4 or 6 or 10 to 12

-   -   or an amino acid sequence in which amino acid residues 1-45,        51-67, 73-109 thereof have at least 90% or 95% identity to the        sequence of amino acid residues 1-45, 51-67, 73-109 respectively        of SEQ ID NO: 4 or 6 or 10 to 12 and in which amino acid        residues 46-50, 68-72 and 110-123 have at least 90% or 95%        identity to the sequence of amino acid residues 46-50, 68-72 and        110-123 respectively of SEQ ID NO: 4 or 6 or 10 to 12.

In the beta chain variable domain, the sequence of

-   -   (i) amino acid residues 1-45 thereof may have (a) at least 90%        identity to the amino acid sequence of residues 1-45 of SEQ ID        NO: 4 or (b) may have one, two or three amino acid residues        inserted or deleted relative to the sequence of (a);    -   (ii) amino acid residues 46-50 may be KGHDR or KGRDR    -   (iii) amino acid residues 51-67 thereof may have (a) at least        90% identity to the sequence of amino acid residues 51-67 of SEQ        ID NO: 4 or (b) may have one, two or three amino acid residues        inserted or deleted relative to the sequence of (a);    -   (iv) amino acid residues 68-72 may be SVFDK;    -   (v) amino acid residues 73-109 thereof may have (a) at least 90%        identity to the sequence of amino acid residues 73-109 of SEQ ID        NO: 4 or (b) may have one, two or three amino acid residues        inserted or deleted relative to the sequence of (a);    -   (vi) amino acids 110-123 is CATSGQGAYNEQFF or CATSGQGAYREQFF or        CATSGQGAYKEQFF

A TCR of the invention may have one of the following combinations ofalpha and beta chain variable domains:

Alpha Chain SEQ ID NO: Beta Chain SEQ ID NO: 3 4 3 6 3 10 3 11 3 12 5 45 6 5 10 5 11 5 12 7 4 7 6 7 10 7 11 7 12 8 4 8 6 8 10 8 11 8 12 9 4 9 69 10 9 11 9 12

Within the scope of the invention are phenotypically silent variants ofany TCR of the invention disclosed herein. As used herein the term“phenotypically silent variants” is understood to refer to a TCR whichincorporates one or more further amino acid changes in addition to thoseset out above which TCR has a similar phenotype to the corresponding TCRwithout said change(s). For the purposes of this application, TCRphenotype comprises antigen binding specificity (K_(D) and/or bindinghalf life) and antigen specificity. A phenotypically silent variant mayhave a K_(D) and/or binding half-life for the GVYDGEEHSV (SEQ ID NO: 2)HLA-A*0201 complex or the GVYDGREHTV (SEQ ID NO: 1) HLA-A*0201 complexwithin 10% of the measured K_(D) and/or binding half-life of thecorresponding TCR without said change(s), when measured under identicalconditions (for example at 25° C. and on the same SPR chip). Suitableconditions are further defined in Example 3. Antigen specificity isfurther defined below. As is known to those skilled in the art, it maybe possible to produce TCRs that incorporate changes in the constantand/or variable domains thereof compared to those detailed above withoutaltering the affinity for the interaction with the GVYDGEEHSV (SEQ IDNO: 2) HLA-A*0201 complex or the GVYDGREHTV (SEQ ID NO: 1) HLA-A*0201complex. In particular, such silent mutations may be incorporated withinparts of the sequence that are known not to be directly involved inantigen binding (e.g. outside the CDRs). Such trivial variants areincluded in the scope of this invention. Those TCRs in which one or moreconservative substitutions have been made also form part of thisinvention.

Mutations can be carried out using any appropriate method including, butnot limited to, those based on polymerase chain reaction (PCR),restriction enzyme-based cloning, or ligation independent cloning (LIC)procedures. These methods are detailed in many of the standard molecularbiology texts. For further details regarding polymerase chain reaction(PCR) and restriction enzyme-based cloning, see Sambrook & Russell,(2001) Molecular Cloning— A Laboratory Manual (3^(rd) Ed.) CSHL Press.Further information on ligation independent cloning (LIC) procedures canbe found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6.

Certain TCRs of the invention have been found to be highly suitable foruse in adoptive therapy. Such TCRs may have a K_(D) for the complex ofless than the 200 μM, for example from about 0.05 μM to about 20 μM orabout 100 μM and/or have a binding half-life (T½) for the complex in therange of from about 0.5 seconds to about 12 minutes. In someembodiments, TCRs of the invention may have a K_(D) for the complex offrom about 0.0511M to about 20 μM, about 0.1 μM to about 5 μM or about0.111M to about 2 μM. Without wishing to be bound by theory, there seemsto be an optimum window of affinity for TCRs with therapeutic use inadoptive cell therapy. Naturally occurring TCRs recognising epitopesfrom tumour antigens are generally of too low affinity (20 microM to 50microM) and very high affinity TCRs (in the nanomolar range or higher)suffer from cross-reactivity issues (Robbins et al (2008) J. Immunol.180 6116-6131; Zhao et al (2007) J. Immunol. 179 5845-5854; Scmid et al(2010) J. Immunol 184 4936-4946).

The TCRs of the invention may be αβ heterodimers or may be in singlechain format. Single chain formats include αβ TCR polypeptides of theVα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-V(3 or Vα-L-Vβ-Cβ types, wherein Vα and Vβ areTCR α and β variable regions respectively, Cα and Cβ are TCR α and βconstant regions respectively, and L is a linker sequence. For use as atargeting agent for delivering therapeutic agents to the antigenpresenting cell the TCR may be in soluble form (i.e. having notransmembrane or cytoplasmic domains). For stability, soluble αβheterodimeric TCRs preferably have an introduced disulfide bond betweenresidues of the respective constant domains, as described, for example,in WO 03/020763. One or both of the constant domains present in an αβheterodimer of the invention may be truncated at the C terminus or Ctermini, for example by up to 15, or up to 10 or up to 8 or fewer aminoacids. For use in adoptive therapy, an αβ heterodimeric TCR may, forexample, be transfected as full length chains having both cytoplasmicand transmembrane domains. TCRs for use in adoptive therapy may containa disulphide bond corresponding to that found in nature between therespective alpha and beta constant domains, additionally oralternatively a non-native disulphide bond may be present.

As will be obvious to those skilled in the art, it may be possible totruncate the sequences provided at the C-terminus and/or N-terminusthereof, by 1, 2, 3, 4, 5 or more residues, without substantiallyaffecting the binding characteristics of the TCR. All such trivialvariants are encompassed by the present invention.

Alpha-beta heterodimeric TCRs of the invention usually comprise an alphachain TRAC constant domain sequence and a beta chain TRBC1 or TRBC2constant domain sequence. The alpha and beta chain constant domainsequences may be modified by truncation or substitution to delete thenative disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2of TRBC1 or TRBC2. The alpha and beta chain constant domain sequencesmay also be modified by substitution of cysteine residues for Thr 48 ofTRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming adisulfide bond between the alpha and beta constant domains of the TCR.

Binding affinity (inversely proportional to the equilibrium constantK_(D)) and binding half-life (expressed as T½) can be determined usingthe Surface Plasmon Resonance (BIAcore) method of Example 3 herein.Measurements may be carried out at 25° C. and at a pH between 7.1 and7.5 using a soluble version of the TCR. It will be appreciated thatdoubling the affinity of a TCR results in halving the K_(D). T½ iscalculated as ln2 divided by the off-rate (k_(off)). So doubling of T½results in a halving in k_(off). K_(D) and k_(off) values for TCRs areusually measured for soluble forms of the TCR, i.e. those forms whichare truncated to remove hydrophobic transmembrane domain residues.Therefore it is to be understood that a given TCR meets the requirementthat it has a binding affinity for, and/or a binding half-life for, theGVYDGREHTV (SEQ ID NO: 1)-HLA-A2 complex and the GVYDGEEHSV (SEQ ID NO:2)-HLA-A2 complex if a soluble form of that TCR meets that requirement.Preferably the binding affinity or binding half-life of a given TCR ismeasured several times, for example 3 or more times, using the sameassay protocol, and an average of the results is taken. The referenceTCR has a K_(D) of approximately 17 μM as measured by that method, andT½ is approximately 1.6S

In a further aspect, the present invention provides nucleic acidencoding a TCR of the invention. In some embodiments, the nucleic acidis cDNA. In some embodiments, the invention provides nucleic acidcomprising a sequence encoding an α chain variable domain of a TCR ofthe invention. In some embodiments, the invention provides nucleic acidcomprising a sequence encoding a (3 chain variable domain of a TCR ofthe invention. The nucleic acid may be non-naturally occurring and/orpurified and/or engineered.

In another aspect, the invention provides a vector which comprisesnucleic acid of the invention. Preferably the vector is a TCR expressionvector.

The invention also provides a cell harbouring a vector of the invention,preferably a TCR expression vector. The vector may comprise nucleic acidof the invention encoding in a single open reading frame, or twodistinct open reading frames, the alpha chain and the beta chainrespectively. Another aspect provides a cell harbouring a firstexpression vector which comprises nucleic acid encoding the alpha chainof a TCR of the invention, and a second expression vector whichcomprises nucleic acid encoding the beta chain of a TCR of theinvention. Such cells are particularly useful in adoptive therapy. Thecells of the invention may be isolated and/or recombinant and/ornon-naturally occurring and/or engineered.

Since the TCRs of the invention have utility in adoptive therapy, theinvention includes a non-naturally occurring and/or purified and/or orengineered cell, especially a T-cell, presenting a TCR of the invention.The invention also provides an expanded population of T cells presentinga TCR of the invention. There are a number of methods suitable for thetransfection of T cells with nucleic acid (such as DNA, cDNA or RNA)encoding the TCRs of the invention (see for example Robbins et al.,(2008) J Immunol. 180: 6116-6131). T cells expressing the TCRs of theinvention will be suitable for use in adoptive therapy-based treatmentof cancer. As will be known to those skilled in the art, there are anumber of suitable methods by which adoptive therapy can be carried out(see for example Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

Soluble TCRs of the invention are useful for delivering detectablelabels or therapeutic agents to the antigen presenting cells and tissuescontaining the antigen presenting cells. The may therefore be associated(covalently or otherwise) with a detectable label (for diagnosticpurposes wherein the TCR is used to detect the presence of cellspresenting the GVYDGEEHSV (SEQ ID NO: 2) or GVYDGREHTV (SEQ ID NO:1)-HLA-A2 complex); a therapeutic agent; or a PK modifying moiety (forexample by PEGylation).

Detectable labels for diagnostic purposes include for instance,fluorescent labels, radiolabels, enzymes, nucleic acid probes andcontrast reagents.

Therapeutic agents which may be associated with the TCRs of theinvention include immunomodulators, radioactive compounds, enzymes(perforin for example) or chemotherapeutic agents (cisplatin forexample). To ensure that toxic effects are exercised in the desiredlocation the toxin could be inside a liposome linked to TCR so that thecompound is released slowly. This will prevent damaging effects duringthe transport in the body and ensure that the toxin has maximum effectafter binding of the TCR to the relevant antigen presenting cells.

Other suitable therapeutic agents include:

-   -   small molecule cytotoxic agents, i.e. compounds with the ability        to kill mammalian cells having a molecular weight of less than        700 Daltons. Such compounds could also contain toxic metals        capable of having a cytotoxic effect. Furthermore, it is to be        understood that these small molecule cytotoxic agents also        include pro-drugs, i.e. compounds that decay or are converted        under physiological conditions to release cytotoxic agents.        Examples of such agents include cis-platin, maytansine        derivatives, rachelmycin, calicheamicin, docetaxel, etoposide,        gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone,        sorfimer sodiumphotofrin II, temozolomide, topotecan,        trimetreate glucuronate, auristatin E vincristine and        doxorubicin;    -   peptide cytotoxins, i.e. proteins or fragments thereof with the        ability to kill mammalian cells. For example, ricin, diphtheria        toxin, pseudomonas bacterial exotoxin A, DNase and RNase;    -   radio-nuclides, i.e. unstable isotopes of elements which decay        with the concurrent emission of one or more of α or β particles,        or grays. For example, iodine 131, rhenium 186, indium 111,        yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213;        chelating agents may be used to facilitate the association of        these radio-nuclides to the high affinity TCRs, or multimers        thereof;    -   immuno-stimulants, i.e. immune effector molecules which        stimulate immune response.

For example, cytokines such as IL-2 and IFN-γ,

-   -   Superantigens and mutants thereof;    -   TCR-HLA fusions;    -   chemokines such as IL-8, platelet factor 4, melanoma growth        stimulatory protein, etc;    -   antibodies or fragments thereof, including anti-T cell or NK        cell determinant antibodies (e.g. anti-CD3, anti-CD28 or        anti-CD16);    -   alternative protein scaffolds with antibody like binding        characteristics    -   complement activators;    -   xenogeneic protein domains, allogeneic protein domains,        viral/bacterial protein domains, viral/bacterial peptides.

One preferred embodiment is provided by a TCR of the inventionassociated (usually by fusion to an N- or C-terminus of the alpha orbeta chain) with an anti-CD3 antibody, or a functional fragment orvariant of said anti-CD3 antibody. Antibody fragments andvariants/analogues which are suitable for use in the compositions andmethods described herein include minibodies, Fab fragments, F(ab′)2fragments, dsFv and scFv fragments, Nanobodies™ (these constructs,marketed by Ablynx (Belgium), comprise synthetic single immunoglobulinvariable heavy domain derived from a camelid (e.g. camel or llama)antibody) and Domain Antibodies (Domantis (Belgium), comprising anaffinity matured single immunoglobulin variable heavy domain orimmunoglobulin variable light domain) or alternative protein scaffoldsthat exhibit antibody like binding characteristics such as Affibodies(Affibody (Sweden), comprising engineered protein A scaffold) orAnticalins (Pieris (German), comprising engineered anticalins) to namebut a few.

For some purposes, the TCRs of the invention may be aggregated into acomplex comprising several TCRs to form a multivalent TCR complex. Thereare a number of human proteins that contain a multimerisation domainthat may be used in the production of multivalent TCR complexes. Forexample the tetramerisation domain of p53 which has been utilised toproduce tetramers of scFv antibody fragments which exhibited increasedserum persistence and significantly reduced off-rate compared to themonomeric scFv fragment. (Willuda et al. (2001) J. Biol. Chem. 276 (17)14385-14392). Haemoglobin also has a tetramerisation domain that couldpotentially be used for this kind of application. A multivalent TCRcomplex of the invention may have enhanced binding capability for theGVYDGREHTV (SEQ ID NO: 1) HLA-A2 complex compared to a non-multimericwild-type or T cell receptor heterodimer of the invention. Thus,multivalent complexes of TCRs of the invention are also included withinthe invention. Such multivalent TCR complexes according to the inventionare particularly useful for tracking or targeting cells presentingparticular antigens in vitro or in vivo, and are also useful asintermediates for the production of further multivalent TCR complexeshaving such uses.

As is well-known in the art, TCRs may be subject to post translationalmodifications. Glycosylation is one such modification, which comprisesthe covalent attachment of oligosaccharide moieties to defined aminoacids in the TCR chain. For example, asparagine residues, orserine/threonine residues are well-known locations for oligosaccharideattachment. The glycosylation status of a particular protein depends ona number of factors, including protein sequence, protein conformationand the availability of certain enzymes. Furthermore, glycosylationstatus (i.e. oligosaccharide type, covalent linkage and total number ofattachments) can influence protein function. Therefore, when producingrecombinant proteins, controlling glycosylation is often desirable.Controlled glycosylation has been used to improve antibody-basedtherapeutics. (Jefferis R., Nat Rev Drug Discov. 2009 March;8(3):226-34.). For soluble TCRs of the invention glycosylation may becontrolled in vivo, by using particular cell lines for example, or invitro, by chemical modification. Such modifications are desirable, sinceglycosylation can improve phamacokinetics, reduce immunogenicity andmore closely mimic a native human protein (Sinclair AM and Elliott S.,Pharm Sci. 2005 August; 94(8):1626-35).

For administration to patients, the TCRs, nucleic acids and/or cells ofthe invention (usually associated with a detectable label or therapeuticagent), may be provided in a pharmaceutical composition together with apharmaceutically acceptable carrier or excipient. Therapeutic or imagingTCRs in accordance with the invention will usually be supplied as partof a sterile, pharmaceutical composition which will normally include apharmaceutically acceptable carrier. This pharmaceutical composition maybe in any suitable form, (depending upon the desired method ofadministering it to a patient). It may be provided in unit dosage form,will generally be provided in a sealed container and may be provided aspart of a kit. Such a kit would normally (although not necessarily)include instructions for use. It may include a plurality of said unitdosage forms.

The pharmaceutical composition may be adapted for administration by anyappropriate route, preferably a parenteral (including subcutaneous,intramuscular, or preferably intravenous) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example bymixing the active ingredient with the carrier(s) or excipient(s) understerile conditions.

Dosages of the substances of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. and a physician willultimately determine appropriate dosages to be used.

TCRs, pharmaceutical compositions, vectors, nucleic acids and cells ofthe invention may be provided in substantially pure form, for example atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% pure.

Also provided by the invention are:

-   -   A TCR, nucleic acid or cell of the invention for use in        medicine, preferably for use in a method of treating cancer,        such as solid tumours (e.g., lung, liver and gastric metastases)        and/or squamous cell carcinomas.    -   the use of a TCR, nucleic acid or cell of the invention in the        manufacture of a medicament for treating cancer.    -   a method of treating cancer in a patient, comprising        administering to the patient a TCR, nucleic acid or cell of the        invention.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

The invention is further described in the following non-limitingexamples.

Reference is made to the enclosed sequences, in which:

-   -   SEQ ID NO: 1 is the MAGE B2 peptide    -   SEQ ID NO: 2 is the MAGE A4 peptide    -   SEQ ID NO: 3 is the amino acid sequence of the extracellular        part of the alpha chain of a parental MAGE-A4-specific TCR, and        SEQ ID NO: 4 shows the amino acid sequence of the extracellular        part of the beta chain of a parental MAGE-A4-specific TCR beta        chain amino acid sequence.    -   SEQ ID NO: 5 shows the amino acid sequence of the alpha chain of        a native Lenti TCR (referred to herein as the “reference TCR”).        The sequence is the same as that of The parental TCR except that        a cysteine is substituted for T162 (i.e. T48 of the TRAC        constant region). SEQ ID NO: 6 is the beta chain of a native        Lenti TCR (referred to herein as the “reference TCR). The        sequence is the same as that of the parental TCR except that a        cysteine is substituted for S169 (i.e. S57 of the TRBC2 constant        region) and A187 is substituted for C187 and D201 is substituted        for N201.    -   SEQ ID NOS: 7, 8 and 9 show the sequences of alpha chains which        may be present in TCRs of the invention. The subsequences        forming the CDR regions, or substantial parts of the CDR        regions, are underlined.    -   SEQ ID NOS: 10-12 show the sequence of the beta chain which may        be present in TCRs of the invention. The subsequences forming        the CDR regions, or substantial parts of the CDR regions are        underlined.    -   SEQ ID NOS: 13-18 show the sequences of alpha and beta chains of        TCRs which could not be improved by the mutation and selection        techniques of the present application.

EXAMPLES Example 1— Cloning of the Reference MAGE-A4 TCR Alpha and BetaChain Variable Region Sequences into pGMT7-Based Expression Plasmids

The parental MAGE-A4 TCR variable alpha and TCR variable beta domains ofSEQ ID NOS: 3 and 4 respectively were cloned into pGMT7-based expressionplasmids containing either Cα or Cβ by standard methods described in(Molecular Cloning a Laboratory Manual Third edition by Sambrook andRussell). Plasmids were sequenced using an Applied Biosystems 3730×1 DNAAnalyzer. The reference MAGE-A4 TCR variable alpha and TCR variable betadomains of SEQ ID NOS: 4 and 5 respectively were cloned in the same way.

The DNA sequence encoding the TCR alpha chain variable region wasligated into pEX956, which was cut with restriction enzymes. The DNAsequence encoding the TCR beta chain variable region was ligated intopEXb21, which was also cut with restriction enzymes.

Ligated plasmids were transformed into competent E. coli strain XL1-bluecells and plated out on LB/agar plates containing 100 μg/mL ampicillin.Following incubation overnight at 37° C., single colonies were pickedand grown in 5 mL LB containing 100 μg/mL ampicillin overnight at 37° C.with shaking. Cloned plasmids were purified using a Miniprep kit(Qiagen) and the plasmids were sequenced using an Applied Biosystems3730×1 DNA Analyzer.

Example 2—Expression, Refolding and Purification of Soluble ReferenceMAGE-A4 TCR

The expression plasmids containing the reference TCR α-chain and β-chainrespectively, as prepared in Example 1, were transformed separately intoE. coli strain BL21pLysS, and single ampicillin-resistant colonies weregrown at 37° C. in TYP (ampicillin 100 μg/ml) medium to OD₆₀₀ of˜0.6-0.8 before inducing protein expression with 0.5 mM IPTG. Cells wereharvested three hours post-induction by centrifugation for 30 minutes at4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25 ml BugBuster (NovaGen) in the presence of MgCl₂ and DNaseI. Inclusion bodypellets were recovered by centrifugation for 30 minutes at 13000 rpm ina Beckman J2-21 centrifuge. Three detergent washes were then carried outto remove cell debris and membrane components. Each time the inclusionbody pellet was homogenised in a Triton buffer (50 mM Tris-HCl pH 8.0,0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA,) before being pelleted bycentrifugation for 15 minutes at 13000 rpm in a Beckman J2-21. Detergentand salt was then removed by a similar wash in the following buffer: 50mM Tris-HCl pH 8.0, 1 mM NaEDTA. Finally, the inclusion bodies weredivided into 30 mg aliquots and frozen at −70° C. Inclusion body proteinyield was quantified by solubilising with 6 M guanidine-HCl and an ODmeasurement was taken on a Hitachi U-2001 Spectrophotometer. The proteinconcentration was then calculated using the extinction coefficient.

Approximately 15 mg of TCR α chain and 15 mg of TCR β chain solubilisedinclusion bodies were thawed from frozen stocks and diluted into 10 mlof a guanidine solution (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT), to ensure complete chaindenaturation. The guanidine solution containing fully reduced anddenatured TCR chains was then injected into 0.5 litre of the followingrefolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5 MUrea. The redox couple (cysteamine hydrochloride and cystaminedihydrochloride) to final concentrations of 6.6 mM and 3.7 mMrespectively, were added approximately 5 minutes before addition of thedenatured TCR chains. The solution was left for ˜30 minutes. Therefolded TCR was dialysed in Spectrapor 1 membrane (Spectrum; ProductNo. 132670) against 10 L H₂O for 18-20 hours. After this time, thedialysis buffer was changed twice to fresh 10 mM Tris pH 8.1 (10 L) anddialysis was continued at 5° C.±3° C. for another ˜8 hours.

Soluble TCR was separated from degradation products and impurities byloading the dialysed refold onto a POROS 50 HQ anion exchange column andeluting bound protein with a gradient of 0-500 mM NaCl in 10 mM Tris pH8.1 over 50 column volumes using an Akta purifier (GE Healthcare). Peakfractions were pooled and a cocktail of protease inhibitors (Calbiochem)were added. The pooled fractions were then stored at 4° C. and analysedby Coomassie-stained SDS-PAGE before being pooled and concentrated.Finally, the soluble TCR was purified and characterised using a GEHealthcare Superdex 75HR gel filtration column pre-equilibrated in PBSbuffer (Sigma). The peak eluting at a relative molecular weight ofapproximately 50 kDa was pooled and concentrated prior tocharacterisation by BIAcore surface plasmon resonance analysis.

Example 3—Binding Characterisation BIAcore Analysis

A surface plasmon resonance biosensor (BIAcore 3000™) can be used toanalyse the binding of a soluble TCR to its peptide-MHC ligand. This isfacilitated by producing soluble biotinylated peptide-HLA (“pHLA”)complexes which can be immobilised to a streptavidin-coated bindingsurface (sensor chip). The sensor chips comprise four individual flowcells which enable simultaneous measurement of T-cell receptor bindingto four different pHLA complexes. Manual injection of pHLA complexallows the precise level of immobilised class I molecules to bemanipulated easily.

Biotinylated class I HLA-A*0201 molecules were refolded in vitro frombacterially-expressed inclusion bodies containing the constituentsubunit proteins and synthetic peptide, followed by purification and invitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). HLA-A*0201-heavy chain was expressed with a C-terminalbiotinylation tag which replaces the transmembrane and cytoplasmicdomains of the protein in an appropriate construct. Inclusion bodyexpression levels of ˜75 mg/litre bacterial culture were obtained. TheMHC light-chain or 132-microglobulin was also expressed as inclusionbodies in E. coli from an appropriate construct, at a level of ˜500mg/litre bacterial culture.

E. coli cells were lysed and inclusion bodies are purified toapproximately 80% purity. Protein from inclusion bodies was denatured in6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mMEDTA, and was refolded at a concentration of 30 mg/litre heavy chain, 30mg/litre β2 m into 0.4 M L-Arginine, 100 mM Tris pH 8.1, 3.7 mMcystamine dihydrochloride, 6.6 mM cysteamine hydrochloride, 4 mg/L ofthe MAGE-A4 GVYDGREHTV (SEQ ID NO: 1) or MAGE-B2 GVYDGEEHSV (SEQ ID NO:2) peptide required to be loaded by the HLA-A*02 molecule, by additionof a single pulse of denatured protein into refold buffer at <5° C.Refolding was allowed to reach completion at 4° C. for at least 1 hour.

Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Twochanges of buffer were necessary to reduce the ionic strength of thesolution sufficiently. The protein solution was then filtered through a1.5 μm cellulose acetate filter and loaded onto a POROS 50HQ anionexchange column (8 ml bed volume). Protein was eluted with a linear0-500 mM NaCl gradient in mM Tris pH 8.1 using an Akta purifier (GEHealthcare). HLA-A*0201-peptide complex eluted at approximately 250 mMNaCl, and peak fractions were collected, a cocktail of proteaseinhibitors (Calbiochem) was added and the fractions were chilled on ice.

Biotin-tagged pHLA molecules were buffer exchanged into 10 mM Tris pH8.1, 5 mM NaCl using a GE Healthcare fast desalting column equilibratedin the same buffer. Immediately upon elution, the protein-containingfractions were chilled on ice and protease inhibitor cocktail(Calbiochem) was added. Biotinylation reagents were then added: 1 mMbiotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl₂, and 5 μg/ml BirAenzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem.266: 9-15). The mixture was then allowed to incubate at room temperatureovernight.

The biotinylated pHLA-A*0201 molecules were purified using gelfiltration chromatography. A GE Healthcare Superdex 75 HR 10/30 columnwas pre-equilibrated with filtered PBS and 1 ml of the biotinylationreaction mixture was loaded and the column was developed with PBS at 0.5ml/min using an Akta purifier (GE Healthcare). Biotinylated pHLA-A*0201molecules eluted as a single peak at approximately 15 ml. Fractionscontaining protein were pooled, chilled on ice, and protease inhibitorcocktail was added. Protein concentration was determined using aCoomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A*01molecules were stored frozen at −20° C.

Such immobilised complexes are capable of binding both T-cell receptorsand the coreceptor CD8αα, both of which may be injected in the solublephase. The pHLA binding properties of soluble TCRs are observed to bequalitatively and quantitatively similar if the TCR is used either inthe soluble or immobilised phase. This is an important control forpartial activity of soluble species and also suggests that biotinylatedpHLA complexes are biologically as active as non-biotinylated complexes.

The BIAcore 3000™ surface plasmon resonance (SPR) biosensor measureschanges in refractive index expressed in response units (RU) near asensor surface within a small flow cell, a principle that can be used todetect receptor ligand interactions and to analyse their affinity andkinetic parameters. The BIAcore experiments were performed at atemperature of 25° C., using PBS buffer (Sigma, pH 7.1-7.5) as therunning buffer and in preparing dilutions of protein samples.Streptavidin was immobilised to the flow cells by standard aminecoupling methods. The pHLA complexes were immobilised via the biotintag. The assay was then performed by passing soluble TCR over thesurfaces of the different flow cells at a constant flow rate, measuringthe SPR response in doing so.

Equilibrium Binding Constant

The above BIAcore analysis methods were used to determine equilibriumbinding constants. Serial dilutions of the disulfide linked solubleheterodimeric form of the reference MAGE-A4 TCR were prepared andinjected at constant flow rate of 5 μl min⁻¹ over two different flowcells; one coated with ˜1000 RU of specific GVYDGREHTV (SEQ ID NO: 1)HLA-A*0201 complex, the second coated with ˜1000 RU of non-specificcomplex. Response was normalised for each concentration using themeasurement from the control cell. Normalised data response was plottedversus concentration of TCR sample and fitted to a non-linear curvefitting model in order to calculate the equilibrium binding constant,K_(D). (Price & Dwek, Principles and Problems in Physical Chemistry forBiochemists (2^(nd) Edition) 1979, Clarendon Press, Oxford). Thedisulfide linked soluble form of the reference MAGE-A4 TCR (Example 2)demonstrated a K_(D) of approximately 2.00 μM. From the same BIAcoredata the T½ was approximately 0.95 s.

Kinetic Parameters

The above BIAcore analysis methods were also used to determineequilibrium binding constants and off-rates.

For high affinity TCRs (see Example 4 below) K_(D) was determined byexperimentally measuring the dissociation rate constant, k_(off), andthe association rate constant, k_(on). The equilibrium constant K_(D)was calculated as k_(off)/k_(on).

TCR was injected over two different cells one coated with ˜1000 RU ofspecific GVYDGREHTV (SEQ ID NO: 1) HLA-A*0201complex, the second coatedwith ˜1000 RU of non-specific complex. Flow rate was set at 50 μl/min.Typically 250 μl of TCR at ˜1 μM concentration was injected. Buffer wasthen flowed over until the response had returned to baseline or >2 hourshad elapsed. Kinetic parameters were calculated using BIAevaluationsoftware. The dissociation phase was fitted to a single exponentialdecay equation enabling calculation of half-life.

Example 4—Preparation of High Affinity TCRs of the Invention

Expression plasmids containing the TCR α-chain and β-chain respectivelywere prepared as in Example 1:

TCR ID Alpha Chain SEQ ID NO: Beta Chain SEQ ID NO: TCR1 (parental) 3 4TCR2 7 4 TCR3 8 4 TCR4 3 9 TCR5 3 10 TCR6 3 11 TCR7 3 12

The plasmids were transformed separately into E. coli strain BL21pLysS,and single ampicillin-resistant colonies grown at 37° C. in TYP(ampicillin 100 μg/ml) medium to OD₆₀₀ of ˜0.6-0.8 before inducingprotein expression with 0.5 mM IPTG. Cells were harvested three hourspost-induction by centrifugation for 30 minutes at 4000 rpm in a BeckmanJ-6B. Cell pellets were lysed with 25 ml Bug Buster (Novagen) in thepresence of MgCl₂ and DNaseI. Inclusion body pellets were recovered bycentrifugation for 30 minutes at 13000 rpm in a Beckman J2-21centrifuge. Three detergent washes were then carried out to remove celldebris and membrane components. Each time the inclusion body pellet washomogenised in a Triton buffer (50 mM Tris-HCl pH 8.0, 0.5% Triton-X100,200 mM NaCl, 10 mM NaEDTA,) before being pelleted by centrifugation for15 minutes at 13000 rpm in a Beckman J2-21. Detergent and salt was thenremoved by a similar wash in the following buffer: 50 mM Tris-HCl pH8.0, 1 mM NaEDTA. Finally, the inclusion bodies were divided into 30 mgaliquots and frozen at −70° C. Inclusion body protein yield wasquantified by solubilising with 6 M guanidine-HCl and an OD measurementwas taken on a Hitachi U-2001 Spectrophotometer. The proteinconcentration was then calculated using the extinction coefficient.

Approximately 10 mg of TCR α chain and 10 mg of TCR β chain solubilisedinclusion bodies for each TCR of the invention were diluted into 10 mlof a guanidine solution (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT), to ensure complete chaindenaturation. The guanidine solution containing fully reduced anddenatured TCR chains was then injected into 0.5 litre of the followingrefolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5 MUrea. The redox couple (cysteamine hydrochloride and cystaminedihydrochloride) to final concentrations of 6.6 mM and 3.7 mMrespectively, were added approximately 5 minutes before addition of thedenatured TCR chains. The solution was left for ˜30 minutes. Therefolded TCR was dialysed in Spectrapor 1 membrane (Spectrum; ProductNo. 132670) against 10 L H₂O for 18-20 hours. After this time, thedialysis buffer was changed twice to fresh 10 mM Tris pH 8.1 (10 L) anddialysis was continued at 5° C.±3° C. for another ˜8 hours.

Soluble TCR was separated from degradation products and impurities byloading the dialysed refold onto a POROS 50HQ anion exchange column andeluting bound protein with a gradient of 0-500 mM NaCl in 10 mM Tris pH8.1 over 15 column volumes using an Akta purifier (GE Healthcare). Thepooled fractions were then stored at 4° C. and analysed byCoomassie-stained SDS-PAGE before being pooled and concentrated.Finally, the soluble TCRs were purified and characterised using a GEHealthcare Superdex 75HR gel filtration column pre-equilibrated in PBSbuffer (Sigma). The peak eluting at a relative molecular weight ofapproximately 50 kDa was pooled and concentrated prior tocharacterisation by BIAcore surface plasmon resonance analysis.

The affinity profiles of the thus-prepared TCRs for the MAGE-A4 epitopeor MAGE-B2 epitope were assessed using the method of Example 3, andcompared with the reference TCR. The results are set forth in thefollowing table:

MAGE A4 K_(D) (μM) MAGE-B2 K_(D) (μM) Reference (TCR1) 65.1 17 TCR2 6.82.1 TCR3 7.6 1.7 TCR4 3.15 0.15 TCR5 14.2 6.4 TCR6 1.349 0.348 TCR7 1.656.227

Attempts were also made to prepare high affinity TCRs based oncombinations of SEQ ID NOS: 13/14, 15/16, 17/18.

In the case of TCR A, which combines alpha chain of SEQ ID NO: 13 andthe Beta chain of SEQ ID NO: 14, cross-reactivity was noted betweenMAGE-A1, MAGE-A10 and PRAME. It was not possible to remove thiscross-reactivity by mutation and selection.

TCR B combines the alpha chain of SEQ ID NO: 15 and the Beta chain ofSEQ ID NO: 16. TCR B could not be folded to form a soluble TCR, so nobinding characterisation was possible.

TCR C combines the alpha chain of SEQ ID NO: 17 and the Beta chain ofSEQ ID NO: 18. This TCR was soluble when expressed and could bid toantigen. However, when expressed in T-cells, TCR C showed no activity.

Example 5— Transfection of T-cells with Parental and Variant MAGE-A4TCRs (a) Lentiviral Vector Preparation by Express-In Mediated TransientTransfection of 293T Cells

A 3rd generation lentiviral packaging system was used to packagelentiviral vectors containing the gene encoding the desired TCR. 293Tcells were transfected with 4 plasmids (one lentiviral vector containingthe TCR alpha chain-P2A-TCR beta chain single ORF gene described inExample 5c (below), and 3 plasmids containing the other componentsnecessary to construct infective but non-replicative lentiviralparticles) using Express-In mediated transfection (Open Biosystems).

For transfection one T150 flask of 293T cells in exponential growthphase was taken, with cells evenly distributed on the plate, andslightly more than 50% confluent. Express-In aliquots were brought toroom temperature. 3 ml Serum-Free Medium (RPMI 1640+10 mM HEPES) wereplaced in a sterile 15 ml conical tube. 174 μl of Express-In Reagentwere added directly into the Serum-Free Medium (this provides for a3.6:1 weight ratio of Reagent to DNA). This was mixed thoroughly byinverting tubes 3-4 times and incubated at room temperature for 5-20minutes.

In a separate 1.5 ml microtube was added 15 μg plasmid DNA to premixedpackaging mix aliquots (containing 18 μg pRSV.REV (Rev expressionplasmid), 18 μg pMDLg/p.RRE (Gag/Pol expression plasmid), 7 μg pVSV-G(VSV glycoprotein expression plasmid), usually ˜22 μl, and pipetted upand down to ensure homogeneity of the DNA mix. Approx 1 mL ofExpress-In/Serum-Free Medium was added to the DNA mix dropwise thenpipetted up and down gently before transferring back to the remainder ofthe Express-In/Serum-Free Medium. The tube was inverted ube 3-4 timesand incubated at room temperature for 15-30 minutes. Old culture mediumwas removed from the flask of cells. Express-In/medium/DNA (3 mL)complex was added directly into the bottom of an upright flask of 293Tcells. Slowly, the flask was placed flat to cover the cells and verygently rocked to ensure even distribution. After 1 minute 22 ml freshculture medium (R10+HEPES: RPMI 1640, 10% heat-inactivated FBS, 1%Pen/Strep/L-glutamine, 10 mM HEPES) was added and the flask carefullyreturned to the incubator. This was incubated overnight at 37° C./5%CO2. After 24 hours, the medium containing packaged lentiviral vectorswas harvested.

To harvest the packaged lentiviral vectors, the cell culture supernatantwas filtered through a 0.45 micron nylon syringe filter, the culturemedium centrifuged at 10,000 g for 18 hours (or 112,000 g for 2 hours),most of the supernatant removed (taking care not to disturb the pellet)and the pellet resuspended in the remaining few mL of supernatant(usually about 2 ml from a 31 ml starting volume per tube). This wassnap frozen on dry ice in 1 ml aliquots and stored at −80° C.

(b) Transduction of T Cells with Packaged Lentiviral Vectors ContainingGene of Interest

Prior to transduction with the packaged lentiviral vectors, human Tcells (CD8 or CD4 or both depending on requirements) were isolated fromthe blood of healthy volunteers. These cells were counted and incubatedovernight in R10 containing 50 U/mL IL-2 at 1×10⁶ cells per ml (0.5mL/well) in 48 well plates with pre-washed anti-CD3/CD28 antibody-coatedmicrobeads (Dynabeads® T cell expander, Invitrogen) at a ratio of 3beads per cell.

After overnight stimulation, 0.5 ml of neat packaged lentiviral vectorwas added to the desired cells. This was incubated at 37° C./5% CO2 for3 days. 3 days post-transduction the cells were counted and diluted to0.5×10⁶ cells/ml. Fresh medium containing IL-2 was added as required.Beads were removed 5-7 days post-transduction. Cells were counted andfresh medium containing IL-2 replaced or added at 2 day intervals. Cellswere kept between 0.5×10⁶ and 1×10⁶ cells/mL. Cells were analysed byflow cytometry from day 3 and used for functional assays (e.g. ELISpotfor IFNγ release, see Example 6) from day 5. From day 10, or when cellsare slowing division and reduced in size, cells are frozen in aliquotsof at least 4×10⁶ cells/vial (at 1×10⁷ cells/ml in 90% FBS/10% DMSO) forstorage.

Example 6— Activation of MAGE B2 TCR Engineered T Cells

The following assay was carried out to demonstrate the activation ofTCR-transduced cytotoxic T lymphocytes (CTLs) in response to tumour celllines. IFN-γ production, as measured using the ELISPOT assay, was usedas a read-out for cytotoxic T lymphocyte (CTL) activation.

ELISPOTs Reagents

Assay media: 10% FCS (Gibco, Cat #2011-09), 88% RPMI 1640 (Gibco, Cat#42401), 1% glutamine (Gibco Cat #25030) and 1% penicillin/streptomycin(Gibco Cat #15070-063).

Wash buffer: 0.01M PBS/0.05% Tween 20

PBS (Gibco Cat #10010)

The Human IFNγ ELISPOT kit (BD Bioscience; Cat #551849) containingcapture and detection antibodies and Human IFN-γ PVDF ELISPOT96 wellplates, with associated AEC substrate set (BD Bioscience, Cat #551951)

Methods Target Cell Preparation

The target cells used in this method were natural epitope-presentingcells: A375 human melanoma cells which are both HLA-A2⁺ MAGE A10⁺.HCT116 human colon cancer, which are HLA-A2⁺ MAGE A10⁻, were used as anegative control. Sufficient target cells (50,000 cells/well) werewashed by centrifugation three times at 1200 rpm, 10 min in a Megafuge®1.0 (Heraeus). Cells were then re-suspended in assay media at 10⁶cells/ml.

Effector Cell Preparation

The effector cells (T cells) used in this method were peripheral bloodlymphocytes (PBL), obtained by negative selection using CD14 and CD25microbead kits (Miltenyi Biotech Cat #130-050-201 and 130-092-983respectively) from freshly isolated peripheral blood mononuclear cells(PBMC) from the venous blood of healthy volunteers. Cells werestimulated with antiCD3/CD28 coated beads (Dynabeads® T cell expander,Invitrogen), transduced with lentivirus carrying the gene encoding thefull αβ TCR of interest (based on the construct described in Example 5)and expanded in assay media containing 50 U/mL IL-2 until between 10 and13 days post transduction. These cells were then placed in assay mediaprior to washing by centrifugation at 1200 rpm, 10 min in a Megafuge®1.0 (Heraeus). Cells were then re-suspended in assay media at a 4× thefinal required concentration.

Plates were prepared as follows: 100 μL anti-IFN-γ capture antibody wasdiluted in 10 ml sterile PBS per plate. 100 μL of the diluted captureantibody was then dispensed into each well. The plates were thenincubated overnight at 4° C. Following incubation the plates were washed(programme 1, plate type 2, Ultrawash Plus 96-well plate washer; Dynex)to remove the capture antibody. Plates were then blocked by adding 200μL of assay media to each well and incubated at room temperature for twohours. The assay media was then washed from the plates (programme 1,plate type 2, Ultrawash Plus 96-well plate washer, Dynex) and anyremaining media was removed by flicking and tapping the ELISPOT plateson a paper towel.

The constituents of the assay were then added to the ELISPOT plate inthe following order:

-   -   50 μL of target cells 10⁶ cells/ml (giving a total of 50,000        target cells/well)    -   50 μL media (assay media)    -   50 μL effector cells (20,000 TCR-transduced PBL cells/well)

The plates were then incubated overnight (37° C./5% CO2). The next daythe plates were washed three times (programme 1, plate type 2, UltrawashPlus 96-well plate washer, Dynex) with wash buffer and tapped dry onpaper towel to remove excess wash buffer. 100 μl of primary detectionantibody was then added to each well. The primary detection antibody wasdiluted into of dilution buffer (the volume required for a single plate)using the dilution specified in the manufacturer's instructions. Plateswere then incubated at room temperature for at least 2 hours prior tobeing washed three times (programme 1, plate type 2, Ultrawash Plus96-well plate washer, Dynex) with wash buffer; excess wash buffer wasremoved by tapping the plate on a paper towel.

Secondary detection was performed by adding 100 μL of dilutedstreptavidin-HRP to each well and incubating the plate at roomtemperature for 1 hour. The streptavidin-HRP was diluted into 10 mLdilution buffer (the volume required for a single plate), using thedilution specified in the manufacturer's instructions. The plates werethen washed three times (programme 1, plate type 2, Ultrawash Plus96-well plate washer, Dynex) with wash buffer and tapped on paper towelto remove excess wash buffer. Plates were then washed twice with PBS byadding 200 μL to each well, flicking the buffer off and tapping on apaper towel to remove excess buffer. No more than 15 min prior to use,one drop (20 uL) of AEC chromogen was added to each 1 ml of AECsubstrate and mixed. 10 ml of this solution was prepared for each plate;100 μL was added per well. The plate was then protected from light usingfoil, and spot development monitored regularly, usually occurring within5-20 min. The plates were washed in tap water to terminate thedevelopment reaction, and shaken dry prior to their disassembly intothree constituent parts. The plates were then allowed to dry at roomtemperature for at least 2 hours prior to counting the spots using anImmunospot® Plate reader (CTL; Cellular Technology Limited).

Example 7—Identification of the Binding Motif by Substitution with AllAlternative Amino Acids

Variants of the native MAGE-B2 peptide were obtained in which the aminoacid residue at each position was sequentially replaced with all 19alternative naturally-occurring amino acid, such that 171 peptides wereprepared in total. The native and amino-acid substituted peptides werepulsed on to antigen presenting cells, and interferon γ (IFNγ)production, as measured using the ELISpot assay, used as a read-out forthe activation of T cells transduced with TCR2. Essential positions weredefined by a greater than 50% reduction in T cell activity relative tothe native peptide.

ELISpot assays were carried as described in Example 6.

The tolerated residues at each position of the peptide are shown below.Underlined amino acids represent the native residue at the correspondingposition in the peptide.

Position Tolerated residues  1 G  2 VI  3 WFY  4 D  5 GN  6CDMAGSETQFKVLIHNR  7 YSWTFQMHPLANGDICE  8 FWVLMAYRKCTIQSHGPN  9VNLPMTKQRIYWSAFEGH 10 FMVAILT

It is therefore apparent that the MAGE B2 TCR2 makes contact with atleast V2 Y3 and D4 of the peptide (SEQ ID NO: 1) when in complex withHLA-A*0201 on the surface of antigen presenting cells.

The invention is further described by the following numbered paragraphs:

-   -   1. A T cell receptor (TCR) having the property of binding to        GVYDGEEHSV (SEQ ID NO: 2) in complex with HLA-A*0201 AND        GVYDGREHTV (SEQ ID NO: 1) in complex with HLA-A*0201 with a        dissociation constant of from about 0.05 μM to about μM when        measured with surface plasmon resonance at 25° C. and at a pH        between 7.1 and 7.5 using a soluble form of the TCR, wherein the        TCR comprises a TCR alpha chain variable domain and a TCR beta        chain variable domain, and wherein the TCR variable domains form        contacts with at least residues V2, Y3 and D4 of GVYDGEEHSV (SEQ        ID NO: 2).    -   2. A TCR according to numbered paragraph 1, which is an        alpha-beta heterodimer, having an alpha chain TRAV10+TRAC        constant domain sequence and a beta chain TRBV24-1+TRBC-2        constant domain sequence.    -   3. A TCR as claimed in numbered paragraph 1, which is in single        chain format of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, or        Vα-L-Vβ-C(3, wherein Vα and VP are TCR α and β variable regions        respectively, Cα and CP are TCR a and β constant regions        respectively, and L is a linker sequence.    -   4. A TCR as claimed in any preceding numbered paragraph, which        is associated with a detectable label, a therapeutic agent or a        PK modifying moiety.    -   5. A TCR as claimed in any preceding numbered paragraph, wherein        the alpha chain variable domain comprises an amino acid sequence        that has at least 80% identity to the sequence of amino acid The        alpha chain variable domain of a TCR of the invention may have        the following mutation:

CDR2 M4 V CDR3 S4 T CDR3 S4 N

-   -   with reference to the numbering shown in SEQ ID NO: 3, and/or    -   the beta chain variable domain may have at least one of the        following mutations:

CDR2 S1 A CDR3 S4 T CDR3 N10 E CDR3 N10 G CDR3 N10 V

-   -   with reference to the numbering shown in SEQ ID NO: 4.    -   6. A TCR as claimed in any preceding numbered paragraph, wherein        the alpha chain variable domain comprises the amino acid        sequence of amino acid residues 1-105 of SEQ ID NO: 3 or 7-9 or    -   an amino acid sequence in which amino acid residues 1-27, 34-47,        and 54-90 thereof have at least 90% or 95% identity to the        sequence of amino acid residues 1-27, 34-47, and 54-90        respectively of SEQ ID NO: 3 or 7-9 and in which amino acid        residues 28-34, 48-53 and 91-105 have at least 90% or 95%        identity to the sequence of amino acid residues 28-33, 48-53 and        91-105 respectively of SEQ ID NO: 3 or 7-9.    -   7. A TCR as claimed in any one of numbered paragraphs 1-7,        wherein the alpha chain variable domain comprises the amino acid        sequence of amino acid residues 1-105 of SEQ ID NO: 7-9 or an        amino acid sequence in which amino acid residues 1-27, 34-47 and        55-89 thereof have at least 90% or 95% identity to the sequence        of amino acid residues 1-27, 34-47, and 55-89 respectively of        SEQ ID NO: 7-9 and in which amino acid residues 28-33, 48-53 and        91-105 have at least 90% or 95% identity to the sequence of        amino acid residues 28-33, 48-53 and 91-105 respectively of SEQ        ID NO: 7-9.    -   8. A TCR as claimed in any preceding numbered paragraph, wherein        in the alpha chain variable domain the sequence of    -   (i) amino acid residues 1-27 thereof has (a) at least 90%        identity to the sequence of amino acid residues 1-27 of SEQ ID        NO: 3 or (b) has one, two or three amino acid residues inserted        or deleted relative to the sequence of (a);    -   (ii) amino acid residues 28-33 of SEQ ID NO: 3;    -   (iii) amino acid residues 34-47 thereof has (a) at least 90%        identity to the sequence of amino acid residues 34-47 of SEQ ID        NO: 3 or (b) has one, two or three amino acid residues inserted        or deleted relative to the sequence of (a);    -   (iv) amino acid residues 48-53 of SEQ ID NO: 3 or amino acid        residues 48 and 50-53 of SEQ ID NO: 3 with amino acid residue 49        of SEQ ID NO: 3 substituted with M in place of T;    -   (v) amino acid residues 54-90 thereof has at least 90% identity        to the sequence of amino acid residues 54-90 of SEQ ID NO: 3 or        has one, two or three insertions, deletions or substitutions        relative thereto;    -   (vi) amino acids 91-105 of SEQ ID NO: 3 or amino acid residues        91-93 and 95-105 of SEQ ID NO: 3 with amino acid residue 94 of        SEQ ID NO: 3 substituted with T instead of S.    -   9. A TCR as claimed in any preceding numbered paragraph, wherein        the beta chain variable domain comprises the amino acid sequence        of SEQ ID NO: 4 or 10-12 or an amino acid sequence in which        amino acid residues 1-45, 51-67, and 73-109 thereof have at        least 90% or 95% identity to the sequence of amino acid residues        1-45, 51-67, and 73-109 respectively of SEQ ID NO: 4 or 10-12        and in which amino acid residues 46-50, 68-72 and 110-123 have        at least 90% or 95% identity to the sequence of amino acid        residues 46-50, 68-72 and 110-123 respectively of SEQ ID NO: 4        or 10-12.    -   10. A TCR according to any preceding numbered paragraph, wherein        in the beta chain variable domain the sequence of    -   (i) amino acid residues 1-45 thereof has (a) at least 90%        identity to the amino acid sequence of residues 1-45 of SEQ ID        NO: 4 or (b) has one, two or three amino acid residues inserted        or deleted relative to the sequence of (a);    -   (ii) amino acid residues 46-50 of SEQ ID NO: 4 or amino acid        residues 46, 47, 49 and 50 of SEQ ID NO: 4 with amino acid        residue 48 of SEQ ID NO: 4 substituted with R instead of H;    -   (iii) amino acid residues 51-67 thereof has (a) at least 90%        identity to the sequence of amino acid residues 51-67 of SEQ ID        NO: 4 or (b) has one, two or three amino acid residues inserted        or deleted relative to the sequence of (a);    -   (iv) amino acid residues 68-73 of SEQ ID NO: 4;    -   (v) amino acid residues 73-109 thereof has (a) at least 90%        identity to the sequence of amino acid residues 73-109 of SEQ ID        NO: 4 or (b) has one, two or three amino acid residues inserted        or deleted relative to the sequence of (a);    -   (vi) amino acid residues 110-123 of SEQ ID NO: 4 or amino acid        residues amino acid residues 110-112 and 114-123 of SEQ ID NO: 4        with amino acid residue 113 of SEQ ID NO: 4 substituted with N        instead of S or amino acid residues 110-118 and 120-123 of SEQ        ID NO: 4 with amino acid residue 19 of SEQ ID NO: 4 substituted        with G instead of N or amino acid residues 110-118 and 120-123        of SEQ ID NO: 4 with amino acid residue 19 of SEQ ID NO: 4        substituted with V instead of N.    -   11. Nucleic acid encoding a TCR as claimed in any one of the        preceding numbered paragraphs.    -   12. An isolated or non-naturally occurring cell, especially a        T-cell, presenting a TCR as claimed in any one of numbered        paragraphs 1 to 10.    -   13. A cell harbouring    -   (a) a TCR expression vector which comprises nucleic acid as        claimed in numbered paragraph 12 in a single open reading frame,        or two distinct open reading frames encoding the alpha chain and        the beta chain respectively; or    -   (b) a first expression vector which comprises nucleic acid        encoding the alpha chain of a TCR as claimed in any of numbered        paragraphs 1 to 11, and a second expression vector which        comprises nucleic acid encoding the beta chain of a TCR as        claimed in any of numbered paragraphs 1 to 11.    -   14. A pharmaceutical composition comprising a TCR as claimed in        any one of numbered paragraphs 1 to 10, nucleic acid of numbered        paragraph 11 or a cell as claimed in numbered paragraph 12 or        numbered paragraph 13, together with one or more        pharmaceutically acceptable carriers or excipients.    -   15. The TCR of any one of numbered paragraphs 1 to 12, nucleic        acid of numbered paragraph 13 or cell of numbered paragraph 14        or numbered paragraph 15 for use in medicine.    -   16. The TCR, nucleic acid or cell for use as claimed in numbered        paragraph 15, for use in a method of treating cancer.

MAGE A4 Epitope SEQ ID NO: 1 GVYDGREHTV MAGE B2 Epitope SEQ ID NO: 2GVYDGEEHSV alpha variable chain SEQ ID NO: 3MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF beta variable chainSEQ ID NO: 4 MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAY NEQFFalpha chain soluble form SEQ ID NO: 5MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSbeta chain soluble form SEQ ID NO: 6MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD mutant alpha variable chain SEQ ID NO: 7MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIVTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFmutant alpha variable chain SEQ ID NO: 8MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTITTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFmutant alpha variable chain SEQ ID NO: 9MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTINTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF mutant beta variable chainSEQ ID NO: 10 MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAY VEQFFmutant beta variable chain SEQ ID NO: 11MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDP GLGLRLIYYAFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATTGQGAY NEQFFmutant beta variable chain SEQ ID NO: 12MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYAFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATTGQGAY EEQFFalpha chain soluble form SEQ ID NO: 13METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVGGYSTLTFGKGTVLLVSPDNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS beta chain soluble form SEQ ID NO: 14MSISLLCCAAFPLLWAGPVNAGVTQTPKFRILKIGQSMTLQCAQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVYFCASSYSRWSPLHFGNGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF alpha chain soluble form SEQ ID NO: 15MQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKMANQAGTALIFGKGTTLSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS beta chain soluble form SEQ ID NO: 16MQDGGITQSPKFQVLRTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASLGGLADEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRADalpha chain soluble form SEQ ID NO: 17MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAERNSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS beta chain soluble formSEQ ID NO: 18 MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLESGVNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

1. A T cell receptor (TCR) that comprises a TCR alpha chain variabledomain and a TCR beta chain variable domain, wherein: (A) the TCR alphachain variable domain is comprised in a sequence in which: (i) aminoacid residues 1-27 has (a) at least 90% identity to the sequence ofamino acid residues 1-27 of SEQ ID No: 3 or (b) one, two or three aminoacid insertions, deletions or substitutions relative to amino acidresidues 1-27 of SEQ ID No: 3; (ii) amino acid residues 28-33 is aminoacid residues 28-33 of SEQ ID NO: 3; (iii) amino acid residues 34-47 has(a) at least 90% identity to the sequence of amino acid residues 34-47of SEQ ID NO: 3 or (b) one, two or three amino acid insertions,deletions or substitutions relative to amino acid residues 34-47 of SEQID No: 3; (iv) amino acid residues 48-53 is amino acid residues 48-53 ofany of SEQ ID NOs: 3 or 7; (v) amino acid residues 54-90 has (a) atleast 90% identity to the sequence of amino acid residues 54-90 of SEQID No: 3 or (b) one, two or three amino acid insertions, deletions orsubstitutions relative to amino acid residues 54-90 of SEQ ID No: 3; and(vi) amino acid residues 91-105 is amino acid residues 91-105 of SEQ IDNO: 3; and (B) the TCR beta chain variable domain is comprised in asequence in which: (i) amino acid residues 1-45 has (a) at least 90%identity to the amino acid sequence of residues 1-45 of SEQ ID No: 4 or(b) one, two or three amino acid insertions, deletions or substitutionsrelative to amino acid residues 1-45 of SEQ ID No: 4; (ii) amino acidresidues 46-50 is amino acid residues 46-50 of SEQ ID NO: 4; (iii) aminoacid residues 51-67 has (a) at least 90% identity to the sequence ofamino acid residues 51-67 of SEQ ID NO: 4 or (b) one, two or three aminoacid insertions, deletions or substitutions relative to amino acidresidues 51-67 of SEQ ID No: 4; (iv) amino acid residues 68-72 is aminoacid residues 68-72 of SEQ ID NO: 4 or 11; (v) amino acid residues73-109 has (a) at least 90% identity to the sequence of amino acidresidues 73-109 of SEQ ID NO: 4 or (b) one, two or three amino acidinsertions, deletions or substitutions relative to amino acid residues73-109 of SEQ ID No: 4; and (vi) amino acid residues 110-123 is aminoacid residues 110-123 of any one of SEQ ID NOs: 10, 11 or 12, or aminoacid residues 110-123 of SEQ ID NO: 4 provided that amino acid residues48-53 of the TCR alpha chain variable domain is amino acid residues48-53 of SEQ ID NO:
 7. 2. The TCR of claim 1, wherein the TCR alphachain variable domain is comprised in the amino acid sequence of SEQ IDNO: 3 or
 7. 3. The TCR of claim 1, wherein the TCR beta chain variabledomain is comprised in the amino acid sequence of any one of SEQ ID NOs:4, 10, 11 or
 12. 4. The TCR of claim 1, wherein the TCR alpha chainvariable domain is comprised in the amino acid sequence of SEQ ID NO: 3or 7, and the TCR beta chain variable domain is comprised in the aminoacid sequence of any one of SEQ ID NOs: 4, 10, 11 or
 12. 5. The TCR ofclaim 1, wherein the TCR alpha chain variable domain is comprised in theamino acid sequence of SEQ ID NO: 7, and the TCR beta chain variabledomain is comprised in the amino acid sequence of SEQ ID NO:
 4. 6. TheTCR of claim 1, wherein the TCR alpha chain variable domain is comprisedin the amino acid sequence of SEQ ID NO: 3, and the TCR beta chainvariable domain is comprised in the amino acid sequence of SEQ ID NO:10.
 7. The TCR of claim 1, wherein the TCR alpha chain variable domainis comprised in the amino acid sequence of SEQ ID NO: 3, and the TCRbeta chain variable domain is comprised in the amino acid sequence ofSEQ ID NO:
 11. 8. The TCR of claim 1, wherein the TCR alpha chainvariable domain is comprised in the amino acid sequence of SEQ ID NO: 3,and the TCR beta chain variable domain is comprised in the amino acidsequence of SEQ ID NO:
 12. 9. The TCR of claim 1, which is an alpha-betaheterodimer, having an alpha chain TRAV10+TRAC constant domain sequenceand a beta chain TRBV24-1+TRBC-2 constant domain sequence.
 10. The TCRof claim 1, which is in single chain format of the type Vα-L-Vβ,Vβ-L-Vα, Vα-Cα-L-Vβ, or Vα-L-Vβ-C(3, wherein Vα and Vβ are TCR α and βvariable regions respectively, Cα and Cβ are TCR a and β constantregions respectively, and L is a linker sequence.
 11. One or morepolynucleotides encoding the TCR of claim
 1. 12. A cell presenting theTCR of claim 1, optionally wherein the cell is isolated or non-naturallyoccurring.
 13. The cell of claim 12, wherein the cell is a T-cell.
 14. Acell comprising the one or more polynucleotides of claim
 11. 15. Anisolated or non-naturally occurring cell presenting a TCR encoded by anucleic acid, wherein the nucleic acid encodes a TCR that comprises: (A)a TCR alpha chain variable domain in which: (i) amino acid residues 1-27has (a) at least 90% identity to the sequence of amino acid residues1-27 of SEQ ID No: 3 or (b) one, two or three amino acid insertions,deletions or substitutions relative to amino acid residues 1-27 of SEQID No: 3; (ii) amino acid residues 28-33 is amino acid residues 28-33 ofSEQ ID NO: 3; (iii) amino acid residues 34-47 has (a) at least 90%identity to the sequence of amino acid residues 34-47 of SEQ ID NO: 3 or(b) one, two or three amino acid insertions, deletions or substitutionsrelative to amino acid residues 34-47 of SEQ ID No: 3; (iv) amino acidresidues 48-53 is amino acid residues 48-53 of any of SEQ ID NOs: 3 or7; (v) amino acid residues 54-90 has (a) at least 90% identity to thesequence of amino acid residues 54-90 of SEQ ID No: 3 or (b) one, two orthree amino acid insertions, deletions or substitutions relative toamino acid residues 54-90 of SEQ ID No: 3; and (vi) amino acid residues91-105 is amino acid residues 91-105 of SEQ ID NO: 3; and (B) the TCRbeta chain variable domain is comprised in a sequence in which: (i)amino acid residues 1-45 has (a) at least 90% identity to the amino acidsequence of residues 1-45 of SEQ ID No: 4 or (b) one, two or three aminoacid insertions, deletions or substitutions relative to amino acidresidues 1-45 of SEQ ID No: 4; (ii) amino acid residues 46-50 is aminoacid residues 46-50 of SEQ ID NO: 4; (iii) amino acid residues 51-67 has(a) at least 90% identity to the sequence of amino acid residues 51-67of SEQ ID NO: 4 or (b) one, two or three amino acid insertions,deletions or substitutions relative to amino acid residues 51-67 of SEQID No: 4; (iv) amino acid residues 68-72 is amino acid residues 68-72 ofSEQ ID NO: 4 or 11; (v) amino acid residues 73-109 has (a) at least 90%identity to the sequence of amino acid residues 73-109 of SEQ ID NO: 4or (b) one, two or three amino acid insertions, deletions orsubstitutions relative to amino acid residues 73-109 of SEQ ID No: 4;and (vi) amino acid residues 110-123 is amino acid residues 110-123 ofany one of SEQ ID NOs: 10, 11 or 12, or amino acid residues 110-123 ofSEQ ID NO: 4, provided that amino acid residues 48-53 of the TCR alphachain variable domain is amino acid residues 48-53 of SEQ ID NO:
 7. 16.The cell of claim 15, wherein the cell is a T-cell.
 17. A pharmaceuticalcomposition comprising the cell of claim 12, together with one or morepharmaceutically acceptable carriers or excipients.
 18. A pharmaceuticalcomposition comprising the cell of claim 15, together with one or morepharmaceutically acceptable carriers or excipients.
 19. A method fortreating cancer in a subject, comprising administering to the subjectthe T cell of claim 13, wherein the subject has a cancer cell thatexpresses a melanoma-associated antigen (MAGE) A4 protein and/or a MAGEB2 protein.
 20. A method for treating cancer in a subject, comprisingadministering to the subject the T cell of claim 16, wherein the subjecthas a cancer cell that expresses a melanoma-associated antigen (MAGE) A4protein and/or a MAGE B2 protein.